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Recent progress in the treatment of advanced melanoma has led to the improved survival of affected patients. However, novel treatments also lead to considerable and distinct skin toxicity. To further characterize cutaneous adverse events (AE) of systemic treatments, we conducted a single-center retrospective study of biopsy-proven cutaneous adverse events of melanoma treatment over a period of 10 years at the University Hospital of Zurich, Switzerland. In 102 identified patients, 135 individual skin AEs developed. Immune checkpoint blockade (ICB) was causal for 81 skin AEs, and 54 were related to targeted therapies (TT). Recorded types of skin AEs included lichenoid, maculopapular, acneiform, urticarial, panniculitis, folliculitis, psoriasiform, granulomatous, eczematous, and others. The incidence of skin AEs was higher with TT (18.54%) than with ICB (9.64%, p = 0.0029). Most AEs were low-grade, although 19.21% of AEs were common terminology criteria for adverse events (CTCAE) Grades 3 or 4. A large spectrum of skin AEs was documented during treatment of advanced melanoma, and distinct phenotypes were observed, depending on treatment classes. AEs occurred earlier during treatment with TT than with ICB, and distinct types of skin AEs were associated with respective treatment classes. This study comprehensively describes skin AEs occurring during systemic treatment for melanoma at a single center.

Background: Primary cutaneous mucinoses (PCM) are rare diseases characterized by dermal or follicular mucin deposits. Objectives: A retrospective study characterizing PCM to compare dermal with follicular mucin to identify its potential origin on a single-cell level. Material and methods: Patients diagnosed with PCM between 2010 and 2020 at our department were included in this study. Biopsy specimens were stained using conventional mucin stains (Alcian blue, PAS) and MUC1 immunohistochemical staining. Multiplex fluorescence staining (MFS) was used to investigate which cells were associated with MUC1 expression in select cases. Results: Thirty-one patients with PCM were included, 14 with follicular mucinosis (FM), 8 with reticular erythematous mucinosis, 2 with scleredema, 6 with pretibial myxedema and one patient with lichen myxedematosus. In all 31 specimens, mucin stained positive for Alcian blue and negative for PAS. In FM, mucin deposition was exclusively found in hair follicles and sebaceous glands. None of the other entities showed mucin deposits in follicular epithelial structures. Using MFS, all cases showed CD4+ and CD8+ T cells, tissue histiocytes, fibroblasts and pan-cytokeratin+ cells. These cells expressed MUC1 at different intensities. MUC1 expression in tissue histiocytes, fibroblasts, CD4+ and CD8+ T cells, and follicular epithelial cells of FM was significantly higher than the same cell types in the dermal mucinoses (p < 0.001). CD8+ T cells were significantly more involved in expression of MUC1 than all other analysed cell types in FM. This finding was also significant in comparison with dermal mucinoses. Conclusion: Various cell types seem to contribute to mucin production in PCM. Using MFS, we showed that CD8+ T cells seem to be more involved in the production of mucin in FM than in dermal mucinoses, which could indicate that mucin in dermal and follicular epithelial mucinoses have different origins.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020 - the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86-98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred, using a phylodynamic model. We found that transmission slowed 35-63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

Metabolic reprogramming is an emerging hallmark of resistance to cancer therapy but may generate vulnerabilities that can be targeted with small molecules. Multi-omics analysis revealed that NRAS-mutated melanoma cells with a mesenchymal transcriptional profile adopt a quiescent metabolic program to resist cellular stress response induced by MEK-inhibitor resistance. However, as a result of elevated baseline ROS levels, these cells become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intra-tumor heterogeneity requires the combination of a ROS-inducer and a MEK-inhibitor to target both tumor growth and metastasis. By ex vivo pharmacoscopy of 62 human metastatic melanomas, we found that MEK-inhibitor resistant tumors significantly benefitted from the combination therapy. Finally, we profiled 486 cancer cell lines and revealed that oxidative stress responses and translational suppression are biomarkers of ROS-inducer sensitivity, independent of cancer indication. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS-inducers in melanoma and other cancers. Statement of Significance Targeted-therapy resistance in cancer arises from genetic selection and both transcriptional and metabolic adaptation. We show that metabolic reprogramming sensitizes resistant cells to ROS-induction in combination with pathway inhibitors. Predictive biomarkers of metabolic sensitivity to ROS-inducing agents were identified in many cancer entities, highlighting the generalizability of this treatment approach.

The exponential increase in algorithm-based mobile health (mHealth) applications (apps) for melanoma screening is a reaction to a growing market. However, the performance of available apps remains to be investigated. In this prospective study, we investigated the diagnostic accuracy of a class 1 CE-certified smartphone app in melanoma risk stratification and its patient and dermatologist satisfaction. Pigmented skin lesions ≥ 3 mm and any suspicious smaller lesions were assessed by the smartphone app SkinVision® (SkinVision® B.V., Amsterdam, the Netherlands, App-Version 6.8.1), 2D FotoFinder ATBM® master (FotoFinder ATBM® Systems GmbH, Bad Birnbach, Germany, Version 3.3.1.0), 3D Vectra® WB360 (Canfield Scientific, Parsippany, NJ, USA, Version 4.7.1) total body photography (TBP) devices, and dermatologists. The high-risk score of the smartphone app was compared with the two gold standards: histological diagnosis, or if not available, the combination of dermatologists’, 2D and 3D risk assessments. A total of 1204 lesions among 114 patients (mean age 59 years; 51% females (55 patients at high-risk for developing a melanoma, 59 melanoma patients)) were included. The smartphone app’s sensitivity, specificity, and area under the receiver operating characteristics (AUROC) varied between 41.3–83.3%, 60.0–82.9%, and 0.62–0.72% according to two study-defined reference standards. Additionally, all patients and dermatologists completed a newly created questionnaire for preference and trust of screening type. The smartphone app was rated as trustworthy by 36% (20/55) of patients at high-risk for melanoma, 49% (29/59) of melanoma patients, and 8.8% (10/114) of dermatologists. Most of the patients rated the 2D TBP imaging (93% (51/55) resp. 88% (52/59)) and the 3D TBP imaging (91% (50/55) resp. 90% (53/59)) as trustworthy. A skin cancer screening by combination of dermatologist and smartphone app was favored by only 1.8% (1/55) resp. 3.4% (2/59) of the patients; no patient preferr

Simple Summary In this study, we investigated the associations of circulating tumor DNA (ctDNA), measured at a random time point during the patient’s treatment, with tumor progression and routine blood markers (protein S100, lactate dehydrogenase (LDH), and C-reactive protein (CRP)) in a cohort of patients with metastatic melanoma. Detectable ctDNA was associated with the presence of extracerebral disease, tumor progression, and poorer overall survival (OS). Elevated S100 and CRP was correlated with detectable ctDNA, whereas LDH was not. Our results further support the use of ctDNA in the clinical management of patients with metastatic melanoma. Abstract Melanoma currently lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Circulating tumor DNA (ctDNA), originating from tumor cells and detectable in plasma, has emerged as a possible biomarker in patients with metastatic melanoma. In this retrospective, single-center study, we collected 129 plasma samples from 79 patients with stage IIIB–IV melanoma as determined by the American Joint Committee on Cancer (AJCC, 8th edition). For the determination of ctDNA levels, we used eight different assays of droplet digital polymerase chain reaction (ddPCR) to detect the most common hotspot mutations in the BRAF and NRAS genes. The aim of the study was to investigate the association of the detectability of ctDNA at a non-prespecified time point in a patient’s treatment with tumor progression, and to correlate ctDNA with commonly used biomarkers (protein S100, LDH, and CRP). Patients with detectable ctDNA progressed more frequently in PET-CT within 12 months than those without detectable ctDNA. Detectability of ctDNA was associated with shorter OS in univariate and multivariate analyses. ctDNA was detectable in a statistically significantly larger proportion of patients with distant metastases (79%) than in patients with no distant metastases or only intracranial metastases (32%). Elevated pr

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions. Keywords: bacterial persistence; gastric cancer; granuloma; mutual interaction of pathogenic bacteria; mycobacterial infection.

Lymphoid neogenesis gives rise to tertiary lymphoid structures (TLS) in the periphery of multiple cancer types including muscle invasive bladder cancer (MIBC) where it has positive prognostic and predictive associations. Here, we explored molecular, clinical, and histological data of The Cancer Genome Atlas, as well as the IMvigor210 dataset to study factors associated with TLS development and function in the tumor microenvironment (TME) of MIBC. We also analyzed tumor immune composition including TLS in an independent, retrospective MIBC cohort. We found that the combination of TLS density and tumor mutational burden provides a novel independent prognostic biomarker in MIBC. Gene expression profiles obtained from intratumoral regions that rarely contain TLS in MIBC showed poor correlation with the prognostic TLS density measured in tumor periphery. Tumors with high TLS density showed increased gene signatures as well as infiltration of activated lymphocytes. Intratumoral B-cell and CD8+ T-cell co-infiltration was frequent in TLS-high samples, and such regions harbored the highest proportion of PD-1+TCF1+ progenitor-like T cells, naïve T cells, and activated B cells when compared to regions predominantly infiltrated by either B cells or CD8+ T cells alone. We found four TLS maturation subtypes; however, differences in TLS composition appeared to be dictated by the TME and not by the TLS maturation status. Finally, we identified one downregulated and three upregulated non-immune cell-related genes in TME with high TLS density, which may represent candidates for tumor-intrinsic regulation of lymphoid neogenesis. Our study provides novel insights into TLS-associated gene expression and immune contexture of MIBC and indicates towards the relevance of B-cell and CD8+ T-cell interactions in anti-tumor immunity within and outside TLS.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes., Funding Agencies|Functional Genomics Center Zurich (FGCZ) [p2155, p2419]; University of Zurich (University Priority Research Program (URPP) Translational Cancer Research); Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [31003A_169859, 310030_192075]; Swiss Cancer Research foundation [KFS-4570-08-2018]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; CancerfondenSwedish Cancer Society [CAN 2018/542]; Candoc Forschungskredit [FK-19-026]

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes., Funding Agencies|Functional Genomics Center Zurich (FGCZ) [p2155, p2419]; University of Zurich (University Priority Research Program (URPP) Translational Cancer Research); Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [31003A_169859, 310030_192075]; Swiss Cancer Research foundation [KFS-4570-08-2018]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; CancerfondenSwedish Cancer Society [CAN 2018/542]; Candoc Forschungskredit [FK-19-026]

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes., Funding Agencies|Functional Genomics Center Zurich (FGCZ) [p2155, p2419]; University of Zurich (University Priority Research Program (URPP) Translational Cancer Research); Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [31003A_169859, 310030_192075]; Swiss Cancer Research foundation [KFS-4570-08-2018]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; CancerfondenSwedish Cancer Society [CAN 2018/542]; Candoc Forschungskredit [FK-19-026]

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes., Funding Agencies|Functional Genomics Center Zurich (FGCZ) [p2155, p2419]; University of Zurich (University Priority Research Program (URPP) Translational Cancer Research); Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [31003A_169859, 310030_192075]; Swiss Cancer Research foundation [KFS-4570-08-2018]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; CancerfondenSwedish Cancer Society [CAN 2018/542]; Candoc Forschungskredit [FK-19-026]

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes., Funding Agencies|Functional Genomics Center Zurich (FGCZ) [p2155, p2419]; University of Zurich (University Priority Research Program (URPP) Translational Cancer Research); Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [31003A_169859, 310030_192075]; Swiss Cancer Research foundation [KFS-4570-08-2018]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; CancerfondenSwedish Cancer Society [CAN 2018/542]; Candoc Forschungskredit [FK-19-026]

Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs), which may result in treatment discontinuation. We sought to describe the onset, frequency, and kinetics of irAEs in melanoma patients in a real-life setting and to further investigate the prognostic role of irAEs in treatment outcomes. In this retrospective single-center cohort study, we included 249 melanoma patients. Onset, grade, and resolution of irAEs and their treatment were analyzed. A total of 191 (74.6%) patients in the non-adjuvant and 65 (25.3%) in the adjuvant treatment setting were identified. In the non-adjuvant setting, 29 patients (59.2%) with anti-CTLA4, 43 (58.1%) with anti-PD1, and 54 (79.4%) with anti-PD1/anti-CTLA4 experienced some grade of irAE and these had an improved outcome. In the adjuvant setting, the frequency of irAEs was 84.6% in anti-CTLA4 and 63.5% in anti-PD1, but no correlation with disease relapse was observed. Patients with underlying autoimmune conditions have a risk of disease exacerbation. Immunomodulatory agents had no impact on treatment efficacy. IrAEs are correlated with increased treatment efficacy in the non-adjuvant setting. Application of steroids and immunomodulatory agents, such as anti-TNF-alpha or anti-IL6, did not affect ICI efficacy. These data support irAEs as possible prognostic markers for ICI treatment.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras$^{Q61K}$; Cdkn2a$^{−/−}$ melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.

BACKGROUND Anti-PD1-based immunotherapy is currently used in most patients with advanced melanoma. Despite the remarkable data regarding overall survival, the optimal treatment duration is still unknown. METHODS We evaluated the outcome of 125 patients with advanced melanoma with and without brain metastases (MBM), treated either with anti-PD1 monotherapy (N = 97) or combined with anti-CTLA4 (N = 28) after elective treatment discontinuation due to complete response (CR) (group A, N = 86), or treatment-limiting toxicity (N = 33) and investigator's decision (ID, N = 6) (group B) with subsequent CR. RESULTS For group A, median duration of treatment (mDoT) was 22 months (range 5-49) and median time to CR 9 months (range 2-47). Accordingly, mDoT for group B was 3 months (range 0-36) and median time to CR 7 months (range 1-32). Seven patients from group A and three from group B experienced disease recurrence. Off-treatment survival was not reached. Median off-treatment response time (mOTRt) was 19 months (range 0-42) and 25 months (range 0-66), respectively. For MBM, mOTRt was 17 months (range 7-41) and 28 months (range 9-39), respectively. After a median follow-up of 38 months (range 9-70), seven (5.6%) patients had deceased, one (0.8%) due to melanoma. CONCLUSIONS Treatment discontinuation is feasible also in patients with MBM. Efficacy outcomes seemed to be similar in both groups of patients who achieved CR, regardless of reason for discontinuation. In patients who experienced disease relapse, treatment re-challenge with anti-PD1 resulted in subsequent renewed response.

BACKGROUND RNA editing is a highly conserved posttranscriptional mechanism that contributes to transcriptome diversity. In mammals, it includes nucleobase deaminations that convert cytidine (C) into uridine (U) and adenosine (A) into inosine (I). Evidence from cancer studies indicates that RNA-editing enzymes promote certain mechanisms of tumorigenesis. On the other hand, recoding editing in mRNA can generate mutations in proteins that can participate in the Major Histocompatibility Complex (MHC) ligandome and can therefore be recognized by the adaptive immune system. Anti-cancer treatment based on the administration of immune checkpoint inhibitors enhance these natural anti-cancer immune responses. RESULTS Based on RNA-Seq datasets, we evaluated the editome of melanoma cell lines generated from patients pre- and post-immunotherapy with immune checkpoint inhibitors. Our results reveal a differential editing in Arthrobacter luteus (Alu) sequences between samples pre-therapy and relapses during therapy with immune checkpoint inhibitors. CONCLUSION These data pave the way towards the development of new diagnostics and therapies targeted to editing that could help in preventing relapses during immunotherapies.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

Background: Melanoma brain metastases (MBM) have a poor prognosis. Systemic treatments that have improved outcomes in advanced melanoma have been shown to have an intracranial (IC) effect. We studied the efficacy and outcomes of combined immune checkpoint inhibitor ipilimumab/nivolumab (Combi-ICI) or targeted therapy (Combi-TT) as first-line treatment in MBM. Methods: MBM patients treated with Combi-ICI or Combi-TT within 3 months after MBM diagnosis. Endpoints were progression-free survival (PFS) and overall survival (OS). Results: 53 patients received Combi-ICI, 32% had symptomatic MBM and 33.9% elevated LDH. 71.7% required local treatment. The disease control rate was 60.3%. IC response rate (RR) was 43.8% at 3-months with durable responses at 6- (46.5%) and 12-months (53.1%). Extracranial (EC) RR was 44.7% at 3-months and 50% at 12-months. Median PFS was 9.6 months (95% CI 3.6-NR) and median overall survival (mOS) 44.8 months (95% CI; 26.2-NR). 63 patients received Combi-TT, 55.6% of patients had symptomatic MBM, 57.2% of patients had elevated LDH and 68.3% of patients required local treatment. The disease control rate was 60.4%. ICRR was 50% at 3-months, but dropped at 6-months (20.9%). ECRR was 69.2% at 3-months and 17.6% at 12-months. Median PFS was 5.8 months (95% CI 4.2-7.6) and mOS 14.2 months (95% CI 8.99-26.8). In BRAFV600 patients, 26.7% of patients received Combi-ICI and 73.3% Combi-TT with OS (p = 0.0053) and mPFS (p = 0.03) in favour to Combi-ICI. Conclusion: Combi-ICI showed prolonged mOS with sustainable IC and EC responses. Despite the initially increased efficacy, Combi-TT responses at 12 months were low. Combi-ICI appeared superior to Combi-TT for OS and PFS in BRAFV600 patients. Other clinical factors are determinants for first-line treatment choice.

Background Despite their low individual metastatic potential, thin melanomas (≤ 1 mm Breslow thickness) contribute significantly to melanoma mortality overall. Therefore, identification of prognostic biomarkers is particularly important in this subgroup of melanoma. Prompted by pre‐clinical results, we investigated cyclin D1 protein and Ki‐67 expression in in situ, metastatic and non‐metastatic thin melanomas. Material and Methods Immunohistochemistry was performed on 112 melanoma specimens, thereof 22 in situ, 48 non‐metastatic and 42 metastatic thin melanomas. Overall, epidermal and dermal cyclin D1 and Ki‐67 expression were semi‐quantitatively evaluated by three independent investigators and compared between groups. Results Epidermal Ki‐67 expression did not differ statistically in in situ and invasive melanoma (P = 0.7). Epidermal cyclin D1 expression was significantly higher in thin invasive than in in situ melanoma (P = 0.003). No difference was found in cyclin D1 expression between metastatic and non‐metastatic invasive tumours. Metastatic and non‐metastatic thin melanomas did not show significant differences in epidermal expression of Ki‐67 and cyclin D1 (P = 0.148 and P = 0.611, respectively). In contrast, strong dermal expression of Ki‐67 was more frequent in metastatic than non‐metastatic samples (28.6 vs. 8.3%, respectively, P = 0.001). The prognostic value of dermal Ki‐67 expression was confirmed by multivariate analysis (P = 0.047). Conclusion We found an increased expression of cyclin D1 invasive thin melanomas compared to in situ melanomas which supports a potential role of this protein in early invasion in melanoma, as suggested by pre‐clinical findings. Moreover, our results confirm that high dermal Ki‐67 expression is associated with an increased risk of metastasis development in thin melanoma and could possibly serve as a prognostic biomarker in clinical practice, especially if combined with additional methods.

Human mesenchymal stromal cell (hMSC) secretomes have shown to influence the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair. The angiogenic potential is of particular interest for the treatment of ischemic diseases. Interestingly, hMSC secretomes isolated from different tissue sources have shown dissimilarities with respect to their angiogenic profile. This study compares angiogenesis of hMSC secretomes from adipose tissue (hADSCs), bone marrow (hBMSCs), and umbilical cord Wharton's jelly (hWJSCs). hMSC secretomes were obtained under xenofree conditions and analyzed by liquid chromatography tandem mass spectrometry (LC/MS-MS). Biological processes related to angiogenesis were found to be enriched in the proteomic profile of hMSC secretomes. hWJSC secretomes revealed a more complete angiogenic network with higher concentrations of angiogenesis related proteins, followed by hBMSC secretomes. hADSC secretomes lacked central angiogenic proteins and expressed most detected proteins to a significantly lower level. In vivo all secretomes induced vascularization of subcutaneously implanted Matrigel plugs in mice. Differences in secretome composition were functionally analyzed with monocyte and endothelial cell (EC) in vitro co-culture experiments using vi-SNE based multidimensional flow cytometry data analysis. Functional responses between hBMSC and hWJSC secretomes were comparable, with significantly higher migration of CD14$^{++}$ CD16$^{-}$ monocytes and enhanced macrophage differentiation compared with hADSC secretomes. Both secretomes also induced a more profound pro-angiogenic phenotype of ECs. These results suggest hWJSCs secretome as the most potent hMSC source for inflammation-mediated angiogenesis induction, while the potency of hADSC secretomes was lowest. This systematic analysis may have implication on the selection of hMSCs for future clinical studies.

Recent progress in the diagnosis and treatment of primary and metastatic cutaneous melanoma (CM) has led to a significant increase in the patients` expectancy of life. The development of additional primary tumors (APT) other than CM represents an important survival issue. Of a total of 1764 CM patients, 80 (4.5%) patients developed APT. For tumors diagnosed after CM, there was a 2.7 fold excess risk for APT compared to the swiss german population. A significantly increased risk was noted for female breast (SIR, 2.46), male larynx (SIR, 76.92), male multiple myeloma (SIR, 11.2), male oesophagus (SIR, 10.8) and thyroid on males (SIR, 58.8) and females (SIR, 38.1). All thyroid cancer cases had a common papillary histological subtype and a high rate of BRAFV600E mutation. Melanoma was the primary cause of death in the vast majority of patients. We used the cancer registry from the Comprehensive Cancer Center Zurich (CCCZ) and retrospectively analyzed patients with CM and APT between 2008 and 2018. We calculated the risk of APT compared to the swiss german population using the standardized incidence ratio (SIR). Patients with CM have an increased risk for hematologic and solid APT. Long-term follow-up is indicated.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4, however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment liquid biopsies from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A ROC curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but on the other hand predicted a poor responses to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

Acral lentiginous melanoma (ALM) is a unique histopathological subtype of melanoma with a poorer prognosis than other cutaneous melanomas. This study aims to evaluate the clinicopathological characteristics, metastatic pattern, prognostic factors, response to systemic therapy, and overall survival (OS) of ALM in a White population. This is a retrospective study of patients who were diagnosed and/or treated for ALM at the Department of Dermatology of the University Hospital Zurich, Switzerland, from January 2005 to December 2015. Overall, 172 patients with histologically confirmed ALM were included in the study. In univariate Cox regression, Breslow thickness (P<0.001), age (P=0.003), status of sentinel lymph node (P=0.005), and ulceration (P=0.008) were identified as significant prognostic factors for OS in ALM. In multivariate analysis, only Breslow thickness (P=0.0003) showed statistical significance. The median OS (mOS) was 155.7 months in the entire cohort (n=172) and 11.2 months for stage IV patients (n=36), irrespective of treatment. When first treatment was considered (n=35), mOS for stage IV patients was 8.9, 16.6, 21.7, and 3.7 months, for patients who had received chemotherapy (ChT) (n=17), immunotherapy (n=9), targeted therapy (TT) (n=3), and no therapy (n=6), respectively. The overall response rate was 44% (7/16 patients) to ChT, 100% to TT (3/3), and 25% to ipilimumab (2/8). In our study, Breslow thickness represents the best prognostic factor for OS. In stage IV ALM patients treated with either immunotherapy or TT, there is a trend for extended mOS compared with ChT.

The recent emergence of cancer immunotherapies initiated a significant shift in the clinical management of metastatic melanoma. Prior to 2011, melanoma patients only had palliative treatment solutions which offered little to no survival benefit. In 2018, with immunotherapy, melanoma patients can now contemplate durable or even complete remission. Treatment with novel immune checkpoint inhibitors, anti-cytotoxic T-lymphocyte protein 4 and anti-programmed cell death protein 1, clearly result in superior median and long-term survivals compared to standard chemotherapy; however, more than half of the patients do not respond to immune checkpoint blockade. Currently, clinicians do not have any effective way to stratify melanoma patients for immunotherapies. Research is now focusing on identifying biomarkers which could predict a patient’s response prior treatment initiation (or very early during treatment course), in order to maximize therapeutic efficacy, avoid unnecessary costs, and undesirable heavy side effects for the patient. Given the rapid developments in this field and the translational potential for some of the biomarkers, we will summarize the current state of biomarker research for immunotherapy in melanoma, with an emphasis on omics technologies such as next-generation sequencing and mass cytometry (CyTOF). Immunotherapy has revolutionized the management of metastatic melanoma. Prior to 2011, the median survival for metastatic melanoma was 9 months, compared to greater than 18 months in 2017 (1). Patients now benefit from novel immune checkpoint inhibitors (ICIs), anti-cytotoxic T-lymphocyte protein 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1). From the latest survival data of the Checkmate 067 trial, progression-free survival (PFS) for ipilimumab is 2.9 months, for nivolumab 6.9 months, and for the combination of nivolumab and ipilimumab 11.5 months. Overall survival (OS) of the ipilimumab group was 19.9 and 37.6 months for the nivolumab group.

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS form in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multi-spectral microscopy, quantitative pathology and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13+ perivascular and CXCL12+LTB+ and PD-L1+ epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Further, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are co-administered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naive LSCC patients treated with corticosteroids before surgery, compared to untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment.

Background: MAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9-12 months. Understanding mechanisms of resistance and identifying effective therapeutic alternatives is one of the most important scientific challenges in melanoma. Using proteomics, we want to specifically gain insight into the pathophysiological process of cerebral metastases. Methods: Cerebral metastases from melanoma patients were initially analyzed by a LC-MS shotgun approach performed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinformatics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocytochemistry was performed. Results: In this pilot study, we were able to identify 5977 proteins by LC-MS analysis (data are available via ProteomeXchange with identifier PXD007592). Based on PFS, samples were classified into good responders (PFS ≥ 6 months) and poor responders (PFS [Formula: see text] 3 months). By evaluating these proteomic profiles according to gene ontology (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifier between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be significantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identified class-discriminating proteins based on nearest shrunken centroids, validated and quantified this signature by a targeted approach and could correlate parts of this signature with resistance using the CPL/MUW proteome database and survival of patients by TCGA analysis. We further validated an EMT-like signature as a major discriminator

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT). We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT). Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases. Overlap of IDO and PDL1 increased after therapy. IT only marginally impacted PDL1 expression over time in contrast to TT. Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

Metastatic melanoma is a highly aggressive disease. Recent progress in immunotherapy (IT) and targeted therapy (TT) has led to significant improvements in response and survival rates in metastatic melanoma patients. The current project aims to determine the benefit of the introduction of these new therapies in advanced melanoma across several regions of Switzerland. This is a retrospective multicenter analysis of 395 advanced melanoma patients treated with standard chemotherapy, checkpoint inhibitors, and kinase inhibitors from January 2008 until December 2014. The 1-year survival was 69% (n=121) in patients treated with checkpoint inhibitors (IT), 50% in patients treated with TTs (n=113), 85% in the IT+TT group (n=66), and 38% in patients treated with standard chemotherapy (n=95). The median overall survival (mOS) from first systemic treatment in the entire study cohort was 16.9 months. mOS of patients treated either with checkpoint or kinase inhibitors (n=300, 14.6 months) between 2008 and 2014 was significantly improved (P<0.0001) compared with patients treated with standard chemotherapy in 2008-2009 (n=95, 7.4 months). mOS of 61 patients with brain metastases at stage IV was 8.1 versus 12.5 months for patients without at stage IV (n=334), therefore being significantly different (P=0.00065). Furthermore, a significant reduction in hospitalization duration compared with chemotherapy was noted. Treatment with checkpoint and kinase inhibitors beyond clinical trials significantly improves the mOS in real life and the results are consistent with published prospective trial data.

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. Experimental Design: PPARg increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals. Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor-sensitive and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. ©2017 AACR.

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity. According to the "phenotype switching" model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state. Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma. CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness. Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression. Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival.

Brain metastases (brain mets) are frequent in metastatic melanoma patients. The aim of this study was to investigate the morphology and progression pattern of brain mets in melanoma patients treated with BRAF inhibitors (BRAFi) compared with patients who did not receive targeted therapy (BRAFi group and control group). The number and size of brain mets were compared between a baseline and a comparative MRI at progression. The number of brain mets was grouped into seven number classes (N=1-4, N=5-10, N=11-20, N=21-30, N=31-40, N=41-50, and N>50) and its difference was reported as the change of class that occurred. The mean size of the newly developed lesions was determined by representative measurements and the evolution of three persisting target lesions was assessed on the basis of modified RECIST criteria. Of 96 patients studied, 42 were in the BRAFi group and 54 were in the control group. Patients under BRAFi treatment had a significantly greater increase in the number of brain mets, where the median change of class for the BRAFi compared with the control group was 2 versus 0 (P<0.01). The mean size of the new lesions was smaller in the BRAFi group. Pre-existing target lesions did not show any prominent or different patterns of how they evolved in either group. Brain mets in patients treated with BRAFi showed a progression pattern characterized by a high propensity to disseminate, which might reflect an in-vivo manifestation of phenotype switching in response to targeted therapy, with a predominance of the invasive/migratory tumor cell phenotype. Drivers of invasiveness may present promising targets for therapeutic interventions.

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.

Cancer including melanoma may be ''addicted" to double strand break (DSB) repair and targeting this process could sensitize them to the lethal effect of DNA damage. PARP1 exerts an important impact on DSB repair as it binds to both single- and double- strand breaks. PARP1 inhibitors might be highly effective drugs triggering synthetic lethality in patients whose tumors have germline or somatic defects in DNA repair genes. We hypothesized that PARP1-dependent synthetic lethality could be induced in melanoma cells displaying downregulation of DSB repair genes. We observed that PARP1 inhibitor olaparib sensitized melanomas with reduced expression of DNA ligase 4 (LIG4) to an alkylatimg agent dacarbazine (DTIC) treatment in vitro, while normal melanocytes remained intact. PARP1 inhibition caused accumulation of DSBs, which was associated with apoptosis in LIG4 deficient melanoma cells. Our hypothesis that olaparib is synthetic lethal with LIG4 deficiency in melanoma cells was supported by selective anti-tumor effects of olaparib used either alone or in combination with dacarbazine (DTIC) in LIG4 deficient, but not LIG4 proficient cells. In addition, olaparib combined with DTIC inhibited the growth of LIG4 deficient human melanoma xenografts. This work for the first time demonstrates the effectiveness of a combination of PARP1 inhibitor olaparib and alkylating agent DTIC for treating LIG4 deficient melanomas. In addition, analysis of the TCGA and transcriptome microarray databases revealed numerous individual melanoma samples potentially displaying specific defects in DSB repair pathways, which may predispose them to synthetic lethality triggered by PARP1 inhibitor combined with a cytotoxic drug.

Increased activity of the epigenetic modifier EZH2 has been associated with different cancers. However, evidence for a functional role of EZH2 in tumorigenesis in vivo remains poor, in particular in metastasizing solid cancers. Here we reveal central roles of EZH2 in promoting growth and metastasis of cutaneous melanoma. In a melanoma mouse model, conditional Ezh2 ablation as much as treatment with the preclinical EZH2 inhibitor GSK503 stabilizes the disease through inhibition of growth and virtually abolishes metastases formation without affecting normal melanocyte biology. Comparably, in human melanoma cells, EZH2 inactivation impairs proliferation and invasiveness, accompanied by re-expression of tumour suppressors connected to increased patient survival. These EZH2 target genes suppress either melanoma growth or metastasis in vivo, revealing the dual function of EZH2 in promoting tumour progression. Thus, EZH2-mediated epigenetic repression is highly relevant especially during advanced melanoma progression, which makes EZH2 a promising target for novel melanoma therapies.

The Cancer Genome Atlas (TCGA) has given researchers and clinicians unprecedented access to many different cancers through multiple platforms that include exome sequencing, comparative genomic hybridisation (CGH) arrays, DNA methylation arrays, RNA sequencing, reverse protein phase arrays (RPPA), and clinical features. Most data are available to the public in their raw and processed forms; however, analysis and interpretation of these data require specialised training and software. To address this problem, online tools such as cBioportal, canEvolve, GDAC firehose, PROGgeneV2, and UCSC Cancer browser have been developed by various groups to explore and perform analyses on the datasets that are easily understandable by basic researchers and clinicians. In this mini-review, we give an overview of the datasets available from TCGA and the public tools available for integrative analysis of survival with the genomic and transcriptomic datasets, and introduce a tool being developed by our group to analyse the datasets within TCGA.

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.

BACKGROUND Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression. RESULTS Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients. CONCLUSIONS Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.

PURPOSE Basal cell carcinomas (BCCs) are tumors ignored by immune surveillance. Activated Hedgehog (Hh) signaling within primary cilia is a key driver in the pathogenesis of BCCs. We examined immune alterations during treatment with systemic Hh inhibitors. EXPERIMENTAL DESIGN We investigated biopsies from patients with BCC before (23 patients) and after 4 weeks of treatment (5 patients) with Hh signaling inhibitor. Ber-Ep4, BCL-2, Ki-67, CD4, CD8, MHC class I, HLA-DR-class II, and SOX9 were analyzed by immunohistochemistry. Primary cilia were analyzed by double immunofluorescence of acetylated tubulin and SOX9. Differential gene expression for 84 cytokines and chemokines was analyzed in 3 patients. RESULTS After 4 weeks of treatment, we found reduction of Ki-67, SOX9, Ber-EP4, and BCL-2 expression in tumors associated with morphologic signs of squamous differentiation. In addition, the number of cilia-positive BCC cells was significantly decreased. An upregulation of MHC I expression on the cell membranes of residual tumor cells and an influx of CD4(+), HLA-DR-class II(+), and CD8(+) cells with invasion into the tumor cell nests were found. Finally, qPCR arrays showed the differential expression of genes involved in modulating immune responses. CONCLUSIONS We show that Hh pathway inhibitor-induced tumor regression is accompanied by a dynamic change of the microenvironment with a disruption of immune privilege involving an influx of cytotoxic T cells, activation of the adaptive immune functions, and a profound alteration of the local chemokine/cytokine network. Clin Cancer Res; 1-9. ©2015 AACR.

Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.

BACKGROUND: Congenital melanocytic nevi (CMNs) are melanocytic neoplasms that can transform into melanoma. However, this development is impeded in the majority of cases and mostly affects patients with large or giant CMNs. METHODS: To elucidate mechanisms that keep CMNs from malignant transformation, CMN tissue biopsies were investigated for p-ERK and senescence markers by immunohistochemistry and for SOX10/CD271 (p75(NTR)) by immunofluorescence. CMN cells were cultivated, and MTT assays were performed for evaluating cell viability. Mutation status for NRAS and BRAF was performed by real-time PCR. RESULTS: 13 CMNs (from patients aged 0.5-11.8 years, mean: 2.7) showed immunoreactivity for SOX10/CD271 (p75(NTR)) in 34.2%. p-ERK was immunoreactive in 80% (4/5); β-galactosidase was significantly stronger expressed in CMNs compared to melanocytic nevi of patients over 70 years (p = 0.0085). The 5 CMN cultures were immunoreactive for SOX10/CD271 (p75(NTR)) in 36.7%. By silencing SOX10 by siRNA in 2 CMN cell cultures, cell viability decreased significantly. NRAS(Q61K) mutation was found in 91.7% (11/12) and BRAF(V600E) in 6.3% of all analyzable CMNs (1/16). CONCLUSIONS: Oncogene-induced senescence might prevent malignant transformation through activation of the mitogen-activated protein kinase pathway. SOX10 is necessary for the viability of human CMN cell cultures and may be responsible for clinical changes during aging.

Background: Congenital melanocytic nevi (CMNs) are melanocytic neoplasms that can transform into melanoma. However, this development is impeded in the majority of cases and mostly affects patients with large or giant CMNs. Methods: To elucidate mechanisms that keep CMNs from malignant transformation, CMN tissue biopsies were investigated for p-ERK and senescence markers by immunohistochemistry and for SOX10/CD271 (p75 NTR ) by immunofluorescence. CMN cells were cultivated, and MTT assays were performed for evaluating cell viability. Mutation status for NRAS and BRAF was performed by real-time PCR. Results: 13 CMNs (from patients aged 0.5–11.8 years, mean: 2.7) showed immunoreactivity for SOX10/ CD271 (p75 NTR ) in 34.2%. p-ERK was immunoreactive in 80% (4/5); β-galactosidase was significantly stronger expressed in CMNs compared to melanocytic nevi of patients over 70 years (p = 0.0085). The 5 CMN cultures were immunoreactive for SOX10/CD271 (p75 NTR ) in 36.7%. By silencing SOX10 by siRNA in 2 CMN cell cultures, cell viability decreased significantly. NRAS Q61K mutation was found in 91.7% (11/12) and BRAF V600E in 6.3% of all analyzable CMNs (1/16). Conclusions: Oncogene-induced senescence might prevent malignant transformation through activation of the mitogen-activated protein kinase pathway. SOX10 is necessary for the viability of human CMN cell cultures and may be responsible for clinical changes during aging.